Hi, I'm Lexi and I'm from Southern California. I love playing golf, playing viola, reading books, and of course science!! Although I am not exactly sure what I want to do when I grow up, I know that it would be something related to biology. I have not decided specifically what field of science I would want to pursue, but I hope that through this camp and with time it will become more clear what I want to do in the future. I'm really looking forward to meeting everyone this summer!
Lignin Degradation for the Production of Biofuels Alexis Kim, Olivia Jerdee, Christina Yoh
Currently, the world relies very heavily on ancient biofuels, also known as fossil fuels, such as gasoline and diesel. Fossil fuels are natural fuels made from decomposed plants and animals that have been buried for millions of years. The problem, however, is that they are not sustainable, and have several negative impacts on the environment.
The burning of ancient biofuels results in byproducts including carbon dioxide and other greenhouse gases. By trapping heat in the atmosphere, these greenhouse gases contribute to global warming, which is upsetting the delicate balance of Earth’s complex ecosystems. The habitats of organisms, especially in cold areas, are being destroyed because of global warming. Additionally, pollution from the burning of biofuels affects organisms in all types of ecosystems.
Fossil fuels are not only harmful to the environment, they are also nonrenewable. The world’s supply of fossil fuels is rapidly decreasing and cannot meet the needs of the growing and developing human population. Though there are claims that we have enough coal to last us millions of years, the truth is that if we increase production to cover our currently depleting coal and oil reserves, it will only last us until 2088.
Biofuels
Consequently, people have been searching for a solution to this inevitable problem, and many have turned to energy derived from more sustainable and renewable resources. This source of energy is biofuels, which are being developed to increase their efficiency and yield. Biofuels are fuels obtained from living matter, and are renewable as they can be derived from crops, which can be grown and harvested in large quantities. There are four categories of biofuels: first generation, second generation, third generation, and fourth generation biofuels.
First generation biofuels are generated from materials that are not green and have harmful effects on the environment, such as high CO2 emissions. Examples of first generation biofuels include fuel created from starch, sugar, or vegetable oil. These fuels also would impact the food supply if they were used in large amounts.
In contrast to first generation fuels, second generation fuels are derived from sources that are more eco-friendly. They naturally occur in greater quantities in the environment, have less of an impact on biodiversity, and produce fewer greenhouse gases. However, many second generation biofuels are not fully developed, and as a result, are not available or efficient enough for regular use. For example, cellulosic ethanol, a second generation biofuel, is derived from inedible parts of plants such as wood, grass, and materials that contribute to the strength of plant cell walls. Another plant called switchgrass is used for biofuel production because it is broadly adaptive, has consistently high yield, requires minimal agricultural input, and is relatively easy to establish from seeds. However, the current issue with producing biofuel from switchgrass is that unlike simple sugars in corn, the cellulose and hemicellulose in switchgrass is embedded in a tough material called lignin, which is difficult to extract.
Another plant called switchgrass is used for biofuel production because it is broadly adaptive, has consistently high yield, requires minimal agricultural input, and is relatively easy to establish from seeds. However, the current issue with producing biofuel from switchgrass is that unlike simple sugars in corn, the cellulose and hemicellulose in switchgrass is embedded in a tough material called lignin, which is difficult to extract.
Lignin is a highly stable and heterogeneous organic polymer that is an important part of the structural material for the cell wall of many vascular plants. It makes up around 30% of the plant biomass, and inhibits maximum extraction of fuel from plants, as it interferes with the utilization of cellulose for production of biofuels. In the environment, lignin normally can only be depolymerized by bacteria and fungi, as they contain the ligninolytic enzymes of manganese peroxidase and lignin peroxidase. With more research and experimentation, switchgrass will show even more promise for future use as fuel.
Algae-derived biofuels are categorized as third generation fuels. This type of fuel produces much more energy per unit than other methods. The problem with algae as a source of biofuel is that it requires large quantities of phosphorus and nitrogen to thrive. As a result, it would take more resources to supply the algae with that nutrition than they can produce. This plant shows promise for future use as fuel, but at the moment, there is difficulty addressing the drawbacks.
As scientists are looking for ways to convert biomass into usable fuel in an efficient way, some have started to bring synthetic biology into the mix. Fourth generation fuels are those that are produced with the aid of synthetic biology. For example, researchers from the DOE’s Joint BioEnergy Institute have engineered strains of E.coli to create biofuel from switchgrass without the help of added enzymes. This greatly reduces the cost of creating this biofuel, as enzymes that break down cellulose and hemicellulose are generally expensive. The scientists did this by altering the bacterium to produce those enzymes that digest the cellulose and hemicellulose into complex sugars, which are then hydrolyzed into simple sugars. The simple sugars are then fermented and produce ethanol, butanol, and isobutanol. These hydrocarbons that have properties of petrochemical fuels and be used in place of diesel, jet fuel, and gasoline.
Biofuels from Synthetic E.Coli
Ethanol from E.Coli
Ethanol is produced from glucose through the fermentative process of pyruvate. Glycolysis converts glucose into pyruvate while supplying ATP for the production of biomass. Under anaerobic conditions, pyruvate can be fermented when the enzyme pyruvate decarboxylase (PDC) converts pyruvate into acetaldehyde, which in turn is converted to ethanol by the enzyme alcohol dehydrogenase (ADH) while losing 1 carbon as CO2. Scientists have started to use synthetic biology to engineer the metabolic pathways in E.Coli to create greater amounts of ethanol by increasing the enzymes related to ethanol production.
This image shows the fermentative process of glucose, which produces ethanol, lactate, and acetate
For instance, professors L.O. Ingram, T.Conway, D.P.Clark, G.W. Sewell, and J.F. Preston conducted research of Genetic Engineering of Ethanol Production in E. Coli (1987). They used enzymes essential to ethanol production and from Zymomonas mobilis, an ethanologenic bacterium, meaning it lacks oxidative phosphorylation and produces ethanol and CO2 and the primary fermentation products, and inserted it into E.coli under the control of a common promoter. The enzymes that were in the pathway were pyruvate decarboxylase, which produces acetaldehyde and CO2, and 2 alcohol dehydrogenase isozymes that break down acetaldehyde into ethanol. These 2 genes were inserted into the E.coli, controlled by a single promoter, creating the new artificial pet operon. The result was increased amounts of alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis which resulted in increased production of ethanol and formed an alternative system for the regeneration of NAD+. ‘
Other scientists have constructed separate metabolic pathways in hopes of gaining the maximum yield of ethanol. Glucose fermentation leads to 2 molecules of acetyl-CoA and 2 molecules of NADH, and the use of pyruvate formate lyase, instead of pyruvate decarboxylase (which causes decarboxylation of pyruvic acid to form acetaldehyde and carbon dioxide), inhibited the E.coli from fermenting like Zymomonas mobilis can. Ingram et. al (1987) transformed the plasmid that carried the pyruvate decarboxylase (PDC) and alcohol dehydrogenase genes (adh B) into E.coli, increasing ethanol production.
This image shows the use of the enzymes pyruvate decarboxylase and alcohol dehydrogenase to result in ethanol
In a more recent experiment, Jay Keasling and his researchers at UC Berkeley basically incorporated genes that enabled E. Coli to directly produce biodiesel, and they used synthetic biology to do this. The researchers cloned genes from Clostridium stercorarium and Bacteroides ovatus (bacteria that thrive in soil & guts of animals) which break down cellulose and then instructed the altered E.coli cells to secrete the enzyme, helping transform the sugar into biodiesel. Higher-chain Alcohols (Eg. Butanol) from E.Coli
However, as ethanol is sometimes not the ideal alternative fuel due to its low energy content (30% less energy than gasoline), scientists have recently been exploring other metabolic pathways that produce desirable fuel-like molecules like butanol and isobutanol in E.Coli. These higher chain alcohols have higher energy contents, and also do not heavily absorb water, making it more useful. The only problem is that Isobutanol/C5 alcohols are not readily produced enough to become a substitute, and need to be further developed to become commercially available.
This image show how isopropanol (an alcohol) is produced from acetyl-coA. Different enzymes were inserted in the pathway to produce different genes to maximize isopropanol production.
James Liao, Shota Atsumi, and Taizo Hanai of UCLA have synthetically engineered the E.coli metabolic pathway to produce alcohols. The alcohols are normally a result of fermentation, but modifying the compound usually results in toxicity of the cell, so the team modified pathways in E.coli instead. As a result, they were able to produce higher-chain alcohols from glucose, a renewable carbon source, including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol. The strategy to do this was essentially using E.coli’s very active amino acid biosynthetic pathway and shifting part of it to produce alcohol.
As shown in the graph, TA11 with pTA39/ pTA36 produced the most isopropanol.
Limitations/Future Advancements
Although many scientists have come up with effective ways to engineer E.coli to extract fuel from biomass, the current issues are that these scientists have not been able to acquire the maximum yield from sugar, and also have to continue optimizing the ionic liquid pretreatment to yield biomass that is easier for E.coli to digest and turn into fuel. In Keasling's case, the synthetic E.Coli have only been able to extract 10% of the possible biofuel. They are trying to aim to find more effective ways to extract 80-90% of the maximum yield out of the biomass to make the E.coli ‘commercially viable’. In addition, there is also a fear that these genetically modified bacteria might escape the lab and start spreading uncontrollably, so Keasling and other scientists are looking for ways to remove key metabolic pathways from E.coli so it would not be able to survive in the wild.
In the case of alcohol production from E.coli, scientists still have a long way to go. According to Chris Somerville, director of Energy Bioscience Institute at UC Berkeley, ethanol is deadly to microbes at a concentration of 14%, and butanol is even more toxic, killing microbes at 2% concentration, which could be a huge struggle to get the yield up and also a possible danger hazard for the environment.
Though engineered E.coli is not yet available commercially and is still readily being improved, the promise of this organism is great - and hopefully, will soon play an important role in producing the fuel of the future.
Our Design
Our solution is to design E. Coli so that it can break down lignin and extract biofuels from it, while also being able to access the cellulose that holds the most potential for ethanol production. This bacteria would be able to survive in salt water, making a single solution be able to be used for the biofuel production as the saltwater and the bacteria would be added simultaneously. We would also design the bacteria to be safer so it could be used commercially.
These are the metabolic pathways of our engineered E.Coli that can break down lignin, while also breaking down the remaining cellulose inside the switchgrass or other plants and converting this to ethanol. The truth tables above show the result with the presence or absence of another substance.
On the side of the diagram, there is a key with the different elements of the pathway, for example, the signal molecule, receptors, promoters, genes, enzymes and more, signaling the process of the pathway. As shown in the image above, there are two separate processes going on in this pathway, one that balances osmotic pressure of the bacterium through the production of glycerol, and one that breaks down lignin, and resulting in sugars that go through an engineered process of alcoholic fermentation.
One pathway takes in glycerol as a receptor molecule and will produce large amounts of glycerol to balance out the osmotic pressure. We will engineer our E.Coli so that it is only able to survive in extremely salty, aquatic environments, which will prevent it from surviving outside of the lab and when it is not supposed to be used. There is very high osmotic pressure that occurs in very salty environments which will cause most bacteria, including unmodified E. coli, to burst.
To balance the osmotic pressure, we will imitate a strategy employed by the Dunaliella salina (a green alga). The Dunaliella salina produces high amounts of glycerol, which enables bacteria to withstand osmotic pressure that occurs in very salty environments We don’t know the exact genes that cause this increased glycerol production, so we have black boxed it. Essentially, our bacteria would produce high amounts of glycerol and will continue to produce more glycerol if the glycerol is not at a set value, signaling a positive feedback loop.
The other pathway breaks down lignin (with salt as a receptor molecule, as the bacteria will be in a salt solution) into sugar, and also break down the remaining cellulose inside the plant (switchgrass) into sugar, which will yield ethanol. This pathway breaks down lignin into sugars, while also taking in the sugar broken down from cellulose, and through the process of alcoholic fermentation, produces increased amounts of ethanol.
Firstly, we have salt as a receptor molecule, because without salt, our bacteria would not be able to break down lignin, as we do not want the salt to randomly break down lignin in the environment when unnecessary. However, we do not have lignin as a receptor molecule so that our bacteria can continue to produce enzymes even when not in contact with lignin, which will allow us to extract ethanol out of lignin more quickly. The presence of salt triggers the peroxidase and laccase operon to produce peroxidase and laccase, enzymes that break down lignin. We found these two enzymes as they are produced by white rot fungi, which are organisms in nature that can actively break down lignin. When salt is present, a signal molecule changes the shape of the promoters of the peroxidase and laccase enzyme genes from the white rot fungi. This allows for RNA Polymerase to transcribe and allows for the production of peroxidase and laccase, which will break down lignin. When salt is not present, RNA Polymerase cannot transcribe the genes and neither are present. The lignin is broken down into CO2 and water by these enzymes. Through the process of photosynthesis, which we have black boxed, we will end up with sugar.
While this is occurring, the cellulose that is in the plant (that is not the lignin) will be treated with cellulase, which will break it down to sugar. The already existing engineered E. coli already produce this enzyme. The sugar becomes a receptor molecule for the promoter for sugar, which will produce pyruvate decarboxylase and alcohol dehydrogenase, which are genes in Z. mobilis that increase ethanol production, and through alcoholic fermentation, we will produce ethanol.
Glucose is a product of photosynthesis, so when photosynthesis takes place, glucose is present. However, glucose can be present even when photosynthesis is not taking place because it is the product of cellulase (the enzyme) breaking down cellulose. The presence of glucose allows for the production of ethanol by fermentation. When glucose is not present, fermentation cannot take place, and therefore, ethanol is not produced.
The presence of glucose, similarly to the presence of salt, activates the genes from Z. mobilis for increased production of alcohol dehydrogenase and pyruvate decarboxylase, which both aid in the fermentation process that results in higher production of ethanol. On the other hand, when glucose is not present, the enzymes for fermentation are not produced and ethanol cannot be generated.
Another feature we are adding to our bacteria is that if there is no salt present, the cell will kill itself. This will help prevent the bacteria from going out of the lab and surviving.
Testing the Design
The goal of our design was to produce ethanol in greater quantities than before in order to increase the efficiency of the production. The first way our design will be tested is to test for the presence and amount of ethanol produced by the modified E. Coli in the saline solution. The saline solution would be distilled so that the ethanol could be separated. Then, the volumes of the water and ethanol would be measured to determine how efficient the bacteria were at producing ethanol.
The second way the design will be checked is by testing for proteins. Our E. Coli cell has genes from the white rot fungi that code for the enzymes manganese peroxidase and laccase, which help to break down lignin, and genes from Z. Mobilis that increase ethanol production during fermentation. A Biuret test would be performed on the saline solution to test for the presence of these proteins. When the design produces higher amounts of ethanol, and tests positive to the Biuret test, then it can be deemed successful.
Conclusion
Our design will improve upon the already existing technology of genetically engineered E. coli that breaks down cellulose into sugar and turns the sugar into ethanol. Our bacteria would break down lignin into CO2 and water, and use photosynthesis to turn those into sugar. We would also allow the bacteria to survive in saltwater, and we would design the bacteria to kill itself if it were not in the presence of salt. Our solution would increase the ethanol yield from switchgrass, and would also make the bacteria less risky to use.
Hi, I'm Lexi and I'm from Southern California. I love playing golf, playing viola, reading books, and of course science!! Although I am not exactly sure what I want to do when I grow up, I know that it would be something related to biology. I have not decided specifically what field of science I would want to pursue, but I hope that through this camp and with time it will become more clear what I want to do in the future. I'm really looking forward to meeting everyone this summer!
Lignin Degradation for the Production of Biofuels
Alexis Kim, Olivia Jerdee, Christina Yoh
The Issue | Biofuels from Synthetic E.Coli | Our Design | | Testing the Design | | Conclusion | Presentation
The Issue
Currently, the world relies very heavily on ancient biofuels, also known as fossil fuels, such as gasoline and diesel. Fossil fuels are natural fuels made from decomposed plants and animals that have been buried for millions of years. The problem, however, is that they are not sustainable, and have several negative impacts on the environment.
The burning of ancient biofuels results in byproducts including carbon dioxide and other greenhouse gases. By trapping heat in the atmosphere, these greenhouse gases contribute to global warming, which is upsetting the delicate balance of Earth’s complex ecosystems. The habitats of organisms, especially in cold areas, are being destroyed because of global warming. Additionally, pollution from the burning of biofuels affects organisms in all types of ecosystems.
Fossil fuels are not only harmful to the environment, they are also nonrenewable. The world’s supply of fossil fuels is rapidly decreasing and cannot meet the needs of the growing and developing human population. Though there are claims that we have enough coal to last us millions of years, the truth is that if we increase production to cover our currently depleting coal and oil reserves, it will only last us until 2088.
Biofuels
Consequently, people have been searching for a solution to this inevitable problem, and many have turned to energy derived from more sustainable and renewable resources. This source of energy is biofuels, which are being developed to increase their efficiency and yield. Biofuels are fuels obtained from living matter, and are renewable as they can be derived from crops, which can be grown and harvested in large quantities. There are four categories of biofuels: first generation, second generation, third generation, and fourth generation biofuels.
First generation biofuels are generated from materials that are not green and have harmful effects on the environment, such as high CO2 emissions. Examples of first generation biofuels include fuel created from starch, sugar, or vegetable oil. These fuels also would impact the food supply if they were used in large amounts.
In contrast to first generation fuels, second generation fuels are derived from sources that are more eco-friendly. They naturally occur in greater quantities in the environment, have less of an impact on biodiversity, and produce fewer greenhouse gases. However, many second generation biofuels are not fully developed, and as a result, are not available or efficient enough for regular use. For example, cellulosic ethanol, a second generation biofuel, is derived from inedible parts of plants such as wood, grass, and materials that contribute to the strength of plant cell walls. Another plant called switchgrass is used for biofuel production because it is broadly adaptive, has consistently high yield, requires minimal agricultural input, and is relatively easy to establish from seeds. However, the current issue with producing biofuel from switchgrass is that unlike simple sugars in corn, the cellulose and hemicellulose in switchgrass is embedded in a tough material called lignin, which is difficult to extract.
Another plant called switchgrass is used for biofuel production because it is broadly adaptive, has consistently high yield, requires minimal agricultural input, and is relatively easy to establish from seeds. However, the current issue with producing biofuel from switchgrass is that unlike simple sugars in corn, the cellulose and hemicellulose in switchgrass is embedded in a tough material called lignin, which is difficult to extract.
Lignin is a highly stable and heterogeneous organic polymer that is an important part of the structural material for the cell wall of many vascular plants. It makes up around 30% of the plant biomass, and inhibits maximum extraction of fuel from plants, as it interferes with the utilization of cellulose for production of biofuels. In the environment, lignin normally can only be depolymerized by bacteria and fungi, as they contain the ligninolytic enzymes of manganese peroxidase and lignin peroxidase. With more research and experimentation, switchgrass will show even more promise for future use as fuel.
Algae-derived biofuels are categorized as third generation fuels. This type of fuel produces much more energy per unit than other methods. The problem with algae as a source of biofuel is that it requires large quantities of phosphorus and nitrogen to thrive. As a result, it would take more resources to supply the algae with that nutrition than they can produce. This plant shows promise for future use as fuel, but at the moment, there is difficulty addressing the drawbacks.
As scientists are looking for ways to convert biomass into usable fuel in an efficient way, some have started to bring synthetic biology into the mix. Fourth generation fuels are those that are produced with the aid of synthetic biology. For example, researchers from the DOE’s Joint BioEnergy Institute have engineered strains of E.coli to create biofuel from switchgrass without the help of added enzymes. This greatly reduces the cost of creating this biofuel, as enzymes that break down cellulose and hemicellulose are generally expensive. The scientists did this by altering the bacterium to produce those enzymes that digest the cellulose and hemicellulose into complex sugars, which are then hydrolyzed into simple sugars. The simple sugars are then fermented and produce ethanol, butanol, and isobutanol. These hydrocarbons that have properties of petrochemical fuels and be used in place of diesel, jet fuel, and gasoline.
Biofuels from Synthetic E.Coli
Ethanol from E.Coli
Ethanol is produced from glucose through the fermentative process of pyruvate. Glycolysis converts glucose into pyruvate while supplying ATP for the production of biomass. Under anaerobic conditions, pyruvate can be fermented when the enzyme pyruvate decarboxylase (PDC) converts pyruvate into acetaldehyde, which in turn is converted to ethanol by the enzyme alcohol dehydrogenase (ADH) while losing 1 carbon as CO2. Scientists have started to use synthetic biology to engineer the metabolic pathways in E.Coli to create greater amounts of ethanol by increasing the enzymes related to ethanol production.
For instance, professors L.O. Ingram, T.Conway, D.P.Clark, G.W. Sewell, and J.F. Preston conducted research of Genetic Engineering of Ethanol Production in E. Coli (1987). They used enzymes essential to ethanol production and from Zymomonas mobilis, an ethanologenic bacterium, meaning it lacks oxidative phosphorylation and produces ethanol and CO2 and the primary fermentation products, and inserted it into E.coli under the control of a common promoter. The enzymes that were in the pathway were pyruvate decarboxylase, which produces acetaldehyde and CO2, and 2 alcohol dehydrogenase isozymes that break down acetaldehyde into ethanol. These 2 genes were inserted into the E.coli, controlled by a single promoter, creating the new artificial pet operon. The result was increased amounts of alcohol dehydrogenase II and pyruvate decarboxylase from Z. mobilis which resulted in increased production of ethanol and formed an alternative system for the regeneration of NAD+. ‘
Other scientists have constructed separate metabolic pathways in hopes of gaining the maximum yield of ethanol. Glucose fermentation leads to 2 molecules of acetyl-CoA and 2 molecules of NADH, and the use of pyruvate formate lyase, instead of pyruvate decarboxylase (which causes decarboxylation of pyruvic acid to form acetaldehyde and carbon dioxide), inhibited the E.coli from fermenting like Zymomonas mobilis can. Ingram et. al (1987) transformed the plasmid that carried the pyruvate decarboxylase (PDC) and alcohol dehydrogenase genes (adh B) into E.coli, increasing ethanol production.
In a more recent experiment, Jay Keasling and his researchers at UC Berkeley basically incorporated genes that enabled E. Coli to directly produce biodiesel, and they used synthetic biology to do this. The researchers cloned genes from Clostridium stercorarium and Bacteroides ovatus (bacteria that thrive in soil & guts of animals) which break down cellulose and then instructed the altered E.coli cells to secrete the enzyme, helping transform the sugar into biodiesel.
Higher-chain Alcohols (Eg. Butanol) from E.Coli
However, as ethanol is sometimes not the ideal alternative fuel due to its low energy content (30% less energy than gasoline), scientists have recently been exploring other metabolic pathways that produce desirable fuel-like molecules like butanol and isobutanol in E.Coli. These higher chain alcohols have higher energy contents, and also do not heavily absorb water, making it more useful. The only problem is that Isobutanol/C5 alcohols are not readily produced enough to become a substitute, and need to be further developed to become commercially available.
James Liao, Shota Atsumi, and Taizo Hanai of UCLA have synthetically engineered the E.coli metabolic pathway to produce alcohols. The alcohols are normally a result of fermentation, but modifying the compound usually results in toxicity of the cell, so the team modified pathways in E.coli instead. As a result, they were able to produce higher-chain alcohols from glucose, a renewable carbon source, including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, and 2-phenylethanol. The strategy to do this was essentially using E.coli’s very active amino acid biosynthetic pathway and shifting part of it to produce alcohol.
Limitations/Future Advancements
Although many scientists have come up with effective ways to engineer E.coli to extract fuel from biomass, the current issues are that these scientists have not been able to acquire the maximum yield from sugar, and also have to continue optimizing the ionic liquid pretreatment to yield biomass that is easier for E.coli to digest and turn into fuel. In Keasling's case, the synthetic E.Coli have only been able to extract 10% of the possible biofuel. They are trying to aim to find more effective ways to extract 80-90% of the maximum yield out of the biomass to make the E.coli ‘commercially viable’. In addition, there is also a fear that these genetically modified bacteria might escape the lab and start spreading uncontrollably, so Keasling and other scientists are looking for ways to remove key metabolic pathways from E.coli so it would not be able to survive in the wild.
In the case of alcohol production from E.coli, scientists still have a long way to go. According to Chris Somerville, director of Energy Bioscience Institute at UC Berkeley, ethanol is deadly to microbes at a concentration of 14%, and butanol is even more toxic, killing microbes at 2% concentration, which could be a huge struggle to get the yield up and also a possible danger hazard for the environment.
Though engineered E.coli is not yet available commercially and is still readily being improved, the promise of this organism is great - and hopefully, will soon play an important role in producing the fuel of the future.
Our Design
Our solution is to design E. Coli so that it can break down lignin and extract biofuels from it, while also being able to access the cellulose that holds the most potential for ethanol production. This bacteria would be able to survive in salt water, making a single solution be able to be used for the biofuel production as the saltwater and the bacteria would be added simultaneously. We would also design the bacteria to be safer so it could be used commercially.
These are the metabolic pathways of our engineered E.Coli that can break down lignin, while also breaking down the remaining cellulose inside the switchgrass or other plants and converting this to ethanol. The truth tables above show the result with the presence or absence of another substance.
On the side of the diagram, there is a key with the different elements of the pathway, for example, the signal molecule, receptors, promoters, genes, enzymes and more, signaling the process of the pathway. As shown in the image above, there are two separate processes going on in this pathway, one that balances osmotic pressure of the bacterium through the production of glycerol, and one that breaks down lignin, and resulting in sugars that go through an engineered process of alcoholic fermentation.
One pathway takes in glycerol as a receptor molecule and will produce large amounts of glycerol to balance out the osmotic pressure. We will engineer our E.Coli so that it is only able to survive in extremely salty, aquatic environments, which will prevent it from surviving outside of the lab and when it is not supposed to be used. There is very high osmotic pressure that occurs in very salty environments which will cause most bacteria, including unmodified E. coli, to burst.
To balance the osmotic pressure, we will imitate a strategy employed by the Dunaliella salina (a green alga). The Dunaliella salina produces high amounts of glycerol, which enables bacteria to withstand osmotic pressure that occurs in very salty environments We don’t know the exact genes that cause this increased glycerol production, so we have black boxed it. Essentially, our bacteria would produce high amounts of glycerol and will continue to produce more glycerol if the glycerol is not at a set value, signaling a positive feedback loop.
The other pathway breaks down lignin (with salt as a receptor molecule, as the bacteria will be in a salt solution) into sugar, and also break down the remaining cellulose inside the plant (switchgrass) into sugar, which will yield ethanol. This pathway breaks down lignin into sugars, while also taking in the sugar broken down from cellulose, and through the process of alcoholic fermentation, produces increased amounts of ethanol.
Firstly, we have salt as a receptor molecule, because without salt, our bacteria would not be able to break down lignin, as we do not want the salt to randomly break down lignin in the environment when unnecessary. However, we do not have lignin as a receptor molecule so that our bacteria can continue to produce enzymes even when not in contact with lignin, which will allow us to extract ethanol out of lignin more quickly. The presence of salt triggers the peroxidase and laccase operon to produce peroxidase and laccase, enzymes that break down lignin. We found these two enzymes as they are produced by white rot fungi, which are organisms in nature that can actively break down lignin. When salt is present, a signal molecule changes the shape of the promoters of the peroxidase and laccase enzyme genes from the white rot fungi. This allows for RNA Polymerase to transcribe and allows for the production of peroxidase and laccase, which will break down lignin. When salt is not present, RNA Polymerase cannot transcribe the genes and neither are present. The lignin is broken down into CO2 and water by these enzymes. Through the process of photosynthesis, which we have black boxed, we will end up with sugar.
While this is occurring, the cellulose that is in the plant (that is not the lignin) will be treated with cellulase, which will break it down to sugar. The already existing engineered E. coli already produce this enzyme. The sugar becomes a receptor molecule for the promoter for sugar, which will produce pyruvate decarboxylase and alcohol dehydrogenase, which are genes in Z. mobilis that increase ethanol production, and through alcoholic fermentation, we will produce ethanol.
Glucose is a product of photosynthesis, so when photosynthesis takes place, glucose is present. However, glucose can be present even when photosynthesis is not taking place because it is the product of cellulase (the enzyme) breaking down cellulose. The presence of glucose allows for the production of ethanol by fermentation. When glucose is not present, fermentation cannot take place, and therefore, ethanol is not produced.
The presence of glucose, similarly to the presence of salt, activates the genes from Z. mobilis for increased production of alcohol dehydrogenase and pyruvate decarboxylase, which both aid in the fermentation process that results in higher production of ethanol. On the other hand, when glucose is not present, the enzymes for fermentation are not produced and ethanol cannot be generated.
Another feature we are adding to our bacteria is that if there is no salt present, the cell will kill itself. This will help prevent the bacteria from going out of the lab and surviving.
Testing the Design
The goal of our design was to produce ethanol in greater quantities than before in order to increase the efficiency of the production. The first way our design will be tested is to test for the presence and amount of ethanol produced by the modified E. Coli in the saline solution. The saline solution would be distilled so that the ethanol could be separated. Then, the volumes of the water and ethanol would be measured to determine how efficient the bacteria were at producing ethanol.
The second way the design will be checked is by testing for proteins. Our E. Coli cell has genes from the white rot fungi that code for the enzymes manganese peroxidase and laccase, which help to break down lignin, and genes from Z. Mobilis that increase ethanol production during fermentation. A Biuret test would be performed on the saline solution to test for the presence of these proteins. When the design produces higher amounts of ethanol, and tests positive to the Biuret test, then it can be deemed successful.
Conclusion
Our design will improve upon the already existing technology of genetically engineered E. coli that breaks down cellulose into sugar and turns the sugar into ethanol. Our bacteria would break down lignin into CO2 and water, and use photosynthesis to turn those into sugar. We would also allow the bacteria to survive in saltwater, and we would design the bacteria to kill itself if it were not in the presence of salt. Our solution would increase the ethanol yield from switchgrass, and would also make the bacteria less risky to use.
Presentation
Google Slides Presentation